The detection and medical/diagnostic assessment of soft-tissue tumors currently requires a battery of relatively sophisticated diagnostic tests. Generally a physician will utilize every appropriate diagnostic test available when cancer is suspected. These tests utilize imaging equipment for a visual, internal examination and laboratory tests on potential tumor cells and secretions to determine the tumor burden. If a tumor is detected and appears to be malignant, a biopsy is performed to arrive at a diagnosis. Only the biopsy is taken as unequivocal evidence of malignancy.
The tests involving imaging equipment can be divided into two basic types: those involving an external energy source, such as X-rays or sound waves, and those involving an internal energy source, such as radioisotopes.
X-ray studies are the most useful tools in staging breast cancer. The method is also responsible for detecting the vast majority of lung cancer cases. Once a suspected site has been identified, precise radiographs can offer valuable information to the physician o the exact location and extent of tumors of the breast or lung. Unfortunately, by tee time the tumor is large enough to be detected by X-rays (1-2 cubic centimeters), the patient's prognosis may be relatively poor. In addition to the relatively low sensitivity of X-rays for soft-tissue tumors, serious concerns continue to be raised about the risks associated with this method's level of radiation exposure.
For breast cancer, the approximate location and size of the tumor can be obtained by ultrasound techniques. Ultrasound provides an image of the tumor from the pattern of echoes arising from high frequency sound waves impinging on the breast. Since ultrasonic examination of large sections of the body would be difficult to interpret and therefore of little value, this procedure is usually employed for breast examinations after a palpatable lump has been detected.
Gallium (Ga-67) citrate is the only radiodiagnostic agent indicated for determining the presence and extent of certain soft-tissue tumors. Gallium has been shown to be of diagnostic utility in tumors of the lung and liver. In this procedure gallium is dosed intravenously; the gallium is then scanned by a gamma camera seeking an enhanced uptake of gallium in tumor tissue.
Gallium scanning suffers from several important drawbacks. The agent is neither tumor nor disease specific. Gallium will not only concentrate in many types of tumors (both benign and malignant) to some extent, but it also will seek out any localized infection. Because of these characteristics, the interpretation of scans obtained with gallium citrate is extremely difficult. The scans usually exhibit low contrast and diffuse areas of radioisotope concentration.
It has been discovered that proteins labeled with a radio-isotope are useful as radiotracers or radioscanners in humans. Examples include radiolabeled exogenous or autologous plasma protein for diagnostics of, e.g., pulmonary embolism; human serum albumin for blood pool imaging; radiolabeled tumor-specific antibodies for soft tumor imaging; radiolabeled enzyme proteins and hormone proteins for diagnosing metabolic and endocrinological disorders. The most widely used radionuclides are iodine-123, iodine-125, iodine-131, indium-111, gallium-67 and technetium-99 m. The iodine isotopes, being a halogen, can irreversibly be incorporated in protein molecules by relatively simple substitution chemistry. The iodine isotopes are less attractive for other reasons, especially the beta radiation emitted by these isotopes and the long half life (8 days) of iodine-131.
Technetium-99 m is generally recognized to be the most desirable radioisotope for radioscanning and radiotracing. Attempts to label proteins with technetium involve either chelating of the technetium ion by chelating groups inherently present in the protein molecule or derivatizing the protein molecules with a chelating group prior to labeling with the technetium. A chelate formed by technetium with chelating groups inherently present in the protein is by its nature not stable enough to prevent exchange of technetium with other protein ligands. Technetium-labeled proteins of this type, therefore, often lack the required biospecificity.
Chelator-derivatized proteins generally involve amino acetic acid compounds as chelators (e.g. ethylenediaminetetraacetic acid (EDTA) or DTPA). Proteins of this kind have been found to form strong chelates with indium-111.
Polyphosphonates, in particular diphosphonates, are generally recognized to be highly desirable ligands for chelating technetium. Prior to this invention proteins derivatized with diphosphonates have not been available. It is therefore an object of this invention to provide diphosphonate-derivatized proteins suitable for chelating technetium-99 m.
It is a further object of this invention to provide a method of labeling the diphosphonate-derivatized proteins utilizing technetium-99 m without denaturization or loss of biological activity of the protein.
It is still a further object of this invention to provide a protein-diphosphonate-technetium chelate, and to provide a method for scintigraphic imaging such as soft tumor imaging, in humans utilizing such chelates.